The field of this invention is the complexes of 53BP2 protein with other proteins, in particular, complexes of 53BP2 with xcex2-tubulin, 53BP2 with p62, 53BP2 with hnRNP G, 53BP2 with 53BP2-IP1, 53BP2 with 53BP2-IP2, and 53BP2 with 53BP2-IP3 proteins. The invention includes antibodies to 53BP2 complexes, and their use in, inter alia, screening, diagnosis, prognosis and therapy. The invention further relates to 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 genes and proteins and derivatives, fragments and analogs, thereof.
The human Bcl2/p53 binding protein, known as 53BP2, or BBP (GenBank Accession Number U58334, Naumovski and Cleary, 1996, Mol. Cell. Biol. 16: 3884-3892) impedes cell cycle progression from G2 to M phase. 53BP2 competes with Bcl2 for binding to p53, and thus is critical for modulation of p53 function (Naumovski and Cleary, 1996, Mol. Cell. Biol. 16: 3884-3892). In turn, p53 is a critical tumor suppressor protein that can activate or repress transcription, and thus mediate cell cycle progression (Murray and Hunt, 1993, The Cell Cycle: An Introduction, W. H. Freeman and Co., New York). 53BP2 binds to the central DNA binding domain of p53 via two adjacent ankryin repeats and an SH3 domain, thus supporting its ability to modulate, inter alia, p53 DNA binding and stability, and thus, its tumor suppression (Iwabuchi et al., 1994, Proc. Natl. Acad. Sci. USA 91: 6098-6102). Importantly, the most frequent p53 mutations observed in human cancers map to the region of 53BP2 binding (Gorina and Pavletich, 1996, Science 274: 1001-1005). 53BP2 also modulates the dephosphorylation status, and thus the function of p53, via its binding to protein phosphatase 1 (PP1), thus inhibiting the latter protein""s ability to dephosphorylate p53 (Helps et al., 1995, FEBS Letts. 377: 295-300). Phosphorylation at multiple p53 sites affects its transcriptional activation/inhibition, but in a complicated fashion that is not easy to predict (Fuchs et al., 1995, Eur J Biochem 228: 625-639; Hecker et al., 1996, Oncogene 12: 953-961). The above data indicate that proteins that interact with 53BP2 have a fairly direct means to modulate p53 function. It has been previously shown that one such protein, PP1, binds to the C-terminal region of 53BP2 containing the critical ankyrin and SH3 binding domains (Helps et al., 1995, FEBS Letts. 377: 295-300). In sum, 53BP2 has likely roles in the control of cell cycle progression, transcriptional regulation, cellular apoptosis and differentiation, intracellular signal transduction, and tumorigenesis.
Human xcex2-tubulin (GenBank Accession No. X79535, Leffers et al., 1994, GenBank direct submission Jun. 1, 1994) exists in at least six different isoforms that are expressed from separate genes in a tissue specific distribution (Ranganathan et al., 1997, Prostate 30: 263-268). Tubulins are critical to the enzymic-mechanical conversion of ATP hydrolysis to molecular movement along microtubules. The C-terminus of xcex2-tubulin, in particular the last 12 amino acid residues, interacts with kinesin motors to modulate microtubule polymerization, dynamics, and drug sensitivity (Ranganathan et al., 1997, Prostate 30: 263-268; Tucker and Goldstein, 1997, J. Biol. Chem. 272: 9481-9488). This function may have pathophysiological significance; type IV xcex2-tubulin is highly expressed in adenocarcinomas of the prostate, and type II xcex2-tubulin expression is up-regulated in adenocarcinomas that become malignant (Ranganathan et al., 1997, Prostate 30: 263-268). Colchicine, which specifically interacts with xcex2-tubulins to arrest cellular outgrowth, is an effective antitumor agent (Banerjee, 1997, Biochem. Biophys. Res. Commun. 231: 698-700). Further, microtubules, possibly through beta-tubulin binding to proteins that contain Src homology 2 (SH2) domains, play important roles in the assembly of signaling molecular complexes involved in cellular transformation (Itoh et al., 1996, J. Biol. Chem. 217: 27931-27935). In summary, xcex2-tubulin has roles in tumorigenesis and tumor progression, cell structure and intracellular protein transport, cell differentiation, and intracellular signalling.
Human p62 (GenBank Accession No. M88108, Wong et al., 1992, Cell 69: 551-558) is a 62 kD tyrosine phosphoprotein that displays significant homology to the hnRNP protein GRP33. p62 associates with the p21wafGTPase-activating protein (GAP). The binding depends on the phosphorylation state of p62 and occurs via SH2 domains in GAP (Wong et al., 1992, Cell 69: 551-558). The protein p62 further associates with Src family tyrosine kinase SH3 domains in signalling proteins. Since p62 can interact with multiple proteins at once via its several SH3 binding domains, p62 serves to physically link Src kinase activity with downstream effectors such as GRB2 and phospholipase C gamma-1 (Richard et al., 1995, Mol. Cell. Biol. 15: 186-197). In its dephosphorylated state, p62 actively binds RNA via a xe2x80x9cKH domainxe2x80x9d (Wang et al., 1995, J. Biol. Chem. 270: 2010-2013). Phosphorylation severely impairs p62 binding to RNA, suggesting that p62 RNA binding is regulated in vivo. p62 is known to specifically interact with ubiquitin via its C-terminal 80 residues (Vadlamudi et al., 1996, J. Biol. Chem. 271: 20235-20237), thus implicating p62 in ubiquitin-mediated proteolysis. It also specifically interacts with CSK, a cytosolic protein tyrosine kinase that negatively regulates Src family protein tyrosine kinases, and it is hypothesized that this binding mediates docking of proteins, including GAP and CSK, to cytoskeletal and membrane regions upon c-Src activation (Neet and Hunter, 1995, Mol. Cell. Biol. 15: 4908-4920). Levels of phospho-p62, detected by Western blotting, increase markedly in v-abl transformed lymphoblasts (a cell model of leukemia) that reach advanced stages of feeder-layer independent agar growth (Clark and Liang, 1995, Leukemia 9: 165-174). In summary, p62 is implicated in cell transformation and tumor progression, intracellular signalling and cellular activation by c-Src, ubiquitin-mediated proteolysis, and mRNA binding and metabolism.
Human hnRNP G protein (GenBank Accession No. Z23064; Soulard et al., 1993, Nucleic Acids Res. 21: 4210-4217) is an RNA binding protein whose homolog (p43) was originally identified as an autoantigenic nuclear protein in dogs with a lupus-like syndrome. It is a glycosylated component of heterogenous nuclear ribonucleoprotein complexes that contains an RNA binding domain at its amino terminus and a carboxyl domain rich in serines, arginines, and glycines (Soulard et al., 1993, Nucleic Acids Res. 21: 4210-4217). Likely roles for hnRNP G include regulation of cell division, translational, and transcription. It may also function in various autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis.
The newly identified 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 proteins are encoded in part by a nucleotide sequence identified as EST R72810 in the GenBank Database (Hillier et al., 1995, GenBank Direct Submission Jun. 2, 1995, Accession No. 157775) obtained from the Soares (human) breast library 2NbHBst. Over a span of 54 nucleotides, the EST R72810 sequence displays 74% identity to the Simian immunodeficiency virus SIVpt5 gene (GenBank Accession No. U05129), but otherwise displays no significant homology to known proteins.
53BP2 complexes with any of xcex2-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 have not been previously described.
Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.
The present invention provides certain compositions and methods of production of protein complexes of 53BP2 with proteins that interact with (i.e., bind to) 53BP2 (the proteins shown to bind with 53BP2 are designated xe2x80x9c53BP2-IPxe2x80x9d for 53BP2 interacting protein, and the complexes of 53BP2 and a 53BP2-IP are designated as 53BP2:53BP2-IP herein). Specifically, the invention relates to complexes of 53BP2, and derivatives, fragments and analogs of 53BP2 with xcex2-tubulin, with p62, with hnRNP G, with 53BP2-IP1, with 53BP2-IP2 and with 53BP2-IP3, and their derivatives, analogs and fragments. The present invention further provides methods of screening for proteins that interact with 53BP2, or derivatives, fragments or analogs, thereof; preferably the method of screening is a yeast two hybrid assay system or a variation thereof.
The invention further relates to nucleotide sequences of 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 genes (human 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 genes and homologs of other species), as well as derivatives (e.g., fragments) and analogs thereof. Nucleic acids hybridizable to or complementary to the foregoing nucleotide sequence, such as the inverse complement (i.e. has the complementary sequence running in reverse orientation to the strand so that the inverse complement would hybridize without mismatches to the nucleic acid strand; thus, for example, where the coding strand is hybridizable to a nucleic acid with no mismatches between the coding strand and the hybridizable strand, then the inverse complement of the hybridizable strand is identical to the coding strand) of the foregoing sequences, are provided. The invention also relates to 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 derivatives and analogs of the invention that are functionally active, i.e., they are capable of displaying one or more known functional activities of a wild-type 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 protein. Such functional activities include, but are not limited to ability to bind with [or compete for binding with] 53BP2, antigenicity [ability to bind (or compete with 53BP2-IP1, 53BP2-IP2 or 53BP2-IP3 for binding) to an anti-53BP2-IP1, anti-53BP2-IP2 or anti-53BP2-IP3 antibody, respectively], and immunogenicity (ability to generate antibody which binds 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3, respectively).
Methods of production of the 53BP2:53BP2-IP complexes and of 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 proteins, and derivatives and analogs of the complexes and proteins, e.g., by recombinant means, are also provided. Pharmaceutical compositions are also provided.
The invention further provides methods of modulating (i.e., inhibiting or enhancing) the activity of 53BP2:53BP2-IP complexes, particularly 53BP2:xcex2-tubulin, 53BP2:p62, 53BP2:hnRNP G, 53BP2:53BP2-IP1, 53BP2:53BP2-IP2, or 53BP2:53BP2-IP3 complexes. The protein components of the complexes have been implicated in cellular functions, including but not limited to: control of cell cycle progression, cellular differentiation and apoptosis, tumorigenesis and tumor progression; regulation of transcription and translation; control of intracellular signal transduction, including c-Src signalling; control of ubiquitin-mediated protein degradation, and processing involving mRNA binding and stability. Accordingly, the invention provides methods of screening 53BP2:53BP2-IP complexes, particularly complexes of 53BP2 with xcex2-tubulin, p62, hnRNP G, 53BP2-IP1, 53BP2-IP2, or 53BP2-IP3 and the 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 proteins, as well as derivatives and analogs of the 53BP2:53BP2-IP complexes and 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 proteins for the ability to alter cell functions, particularly those cell functions in which 53BP2 and/or a 53BP2-IP has been implicated, such as but not limited to, cell proliferation, differentiation and apoptosis, tumorigenesis and cell transformation, intracellular signal transduction, gene expression, ubiquitin-mediated protein degradation, and mRNA stability.
The present invention also relates to therapeutic and prophylactic as well as diagnostic, prognostic, and screening methods and compositions based upon 53BP2:53BP2-IP complexes (and the nucleic acids encoding the individual proteins that participate in the complexes) as well as 53BP2-IP1 and 53BP2-IP2 and 53BP2-IP3 proteins and nucleic acids. Therapeutic compounds of the invention include, but are not limited to, 53BP2:53BP2-IP complexes and complexes where one or both members of the complex is a derivative or analog of 53BP2 or 53BP2-IP; 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 proteins and derivatives, fragments and analogs thereof; antibodies to and nucleic acids encoding the foregoing; and antisense nucleic acids to the nucleotide sequences encoding the complex components and 53BP2-IP1, 53BP2-IP2, and 53BP2-IP3 antisense nucleic acids. Diagnostic, prognostic and screening kits are also provided.
Animal models and methods of screening for modulators (i.e. agonists, antagonists and inhibitors) of the activity of 53BP2:53BP2-IP complexes and 53BP2-IP1, 53BP2-IP2 and 53BP2-IP3 proteins are also provided.
Methods of identifying molecules that inhibit, or alternatively, that increase formation of 53BP2:53BP2-IP complexes are also provided.